JP2000217574A - Method for applying reagent on immobilized biological material so as to target the material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of applying at least one kind of a reagent on an immobilized biological material so as to target the material. SOLUTION: This method comprises (a) a step for localizing immobilized biological materials to be examined, and recording the positions, (b) a step for positioning a tool for applying a reagent at the position of the immobilized biological material, recorded at the step (a), and (c) a step for applying the reagent on the immobilized biological material. One or several positions of the same or different biological materials immobilized on a carrier are recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for targeting and applying at least one reagent on an immobilized biological material, and to at least one reagent immobilized thereon for contact hybridization. Diagnostic kit comprising a small area local cover.

[0002]

BACKGROUND OF THE INVENTION Modern methods for examining fixed biological material, such as cells or chromosomes, often use expensive reagents, for example, where fluorescent dyes are used for labeling. Is done. Examples of such methods are, inter alia, FISH (fluorescence in situ hybridization)
And multicolor FISH, where cells are hybridized, for example, with a fluorescently labeled nucleic acid probe or probe mixture. An example of the application of such a FISH method is a mid-term analysis. Since metaphase chromosomes form only a small part of the material to be hybridized and the region to be hybridized, only a small part of the DNA probe used can be used for this analysis. Most of the DNA probes therefore remain unused, washed away and discarded.

[0003]

A method for avoiding this situation consists in targeting and applying reagents on a region of the carrier, such as, for example, a microscope slide, on which the slides are of interest. Biological material, in the case of metaphase analysis, the metaphase chromosomes are thus fixed, for example by targeted dripping of a moderately small amount of liquid. In this way, for example, expensive initial materials of reagents, such as fluorescently labeled DNA probes, can be maximally utilized.

The technical problem of the present invention is therefore to provide a method for targeting (specifically) applying at least one reagent onto an immobilized biological material, said method comprising the steps of: Allows improved utilization of applied reagents.

[0005]

This problem is solved by the embodiments of the invention as set forth in the claims.

In particular, there is provided a method for targeting and applying at least one reagent on a biological material immobilized on a carrier, the method comprising the steps of (a) immobilizing the biological material to be investigated Localizing (finding) and recording its position,
(b) positioning the device for applying at least one reagent on the location of the immobilized biological material recorded in step (a); and (c) at least positioning the device on the immobilized biological material. Applying one type of reagent, wherein one or several locations of the same or different immobilized biological material on the carrier are recorded.

The expression "biological material" can include all naturally occurring materials, especially cells, cellular components such as chromosomes, especially metaphase chromosomes. For example, a biological material such as a cell or chromosome may have one or several specific markers, for example, surface markers such as labeled antibodies,
Alternatively, it can be labeled with 23 different distinguishable labeled probes for chromosome analysis. The expression “carrier” can include all objects on which biological material can be retained, in particular microscope slides. The expression "immobilized" means that the biological material is tethered and / or immobilized on the carrier using methods known to those skilled in the art.

In step (a) of the method according to the invention, the localization of the immobilized biological material to be investigated may comprise a scanning device or a microscope lens, for example an aiming device such as a search optic. Scanning
ning). In a preferred embodiment of the method of the present invention, the search optics in step (b) is replaced by an instrument suitable for applying at least one reagent. According to a preferred embodiment of the method according to the invention, one or several photographs of the localized immobilized biological material are provided during step (a) by a scanning device and an image processing unit, for example a CCD (charge It can be recorded in combination with a (coupled element) camera. The photographs can preferably be displayed together to pre-select the appropriate fixed biological material. The recording of the position of the immobilized biological material is performed, for example, using a position scanning device on a microscope slide, preferably on a digital storage medium, such as a diskette, hard disk, removable disk unit, or a compact disk, etc. It can be performed in combination with computer-assisted recording / processing and storage of immobilized biological material location data on a physical storage medium.

For applying at least one reagent,
The instrument (preferably automated) may include a pipette, for example, a microliter pipette.

In a preferred embodiment of the present invention, the method of targeting and applying reagents on immobilized cells or cell components further comprises: (d) providing a small area local cover, the location of which is determined in step (a). Positioning the applied instrument over the location of the immobilized biological material recorded in step.

[0011] A small area local cover may include, for example, a microcover slip.

[0012]

According to a preferred embodiment of the method according to the invention, the application of the at least one reagent and / or the application of a small-area local cover is performed automatically. In a further preferred embodiment of the method of the invention, the small area topical cover is coated or moistened with a reagent.

According to a preferred embodiment of the method of the invention,
At least one intermediate step for treating, preferably washing, the immobilized biological material comprises the localization of the immobilized biological material to be examined in step (a) and its location record, and the step (b) )) And the positioning of the instrument applying the at least one reagent onto the position recorded in step (a) of the immobilized biological material.

According to a further embodiment of the method of the invention,
The evaluation is carried out after step (c) or (d), for example for analytical scanning, including scanning devices, or for directed scanning, including, for example, photometric and / or chemiluminescent measuring devices such as fluorescence measurements. Preferably, the device is performed by positioning it on the position of the immobilized biological material whose position was recorded in step (a).

According to a further embodiment of the method of the invention,
The at least one reagent comprises a nucleic acid selected from DNA or RNA, or comprises a polypeptide. The expression "nucleic acid" refers to deoxyribonucleotides and / or
Or natural, semi-synthetic or modified nucleic acid molecules of ribonucleotides, and / or aminonucleotides or
(α-S) -Nucleotide means a modified nucleotide such as triphosphate. Polypeptides can include, for example, proteins such as antibodies, which can be polyclonal or monoclonal.

According to a further embodiment of the method of the invention,
At least one reagent is provided with one or several labels. The expression “label” is incorporated into or attached to at least one molecule of the reagent.
A suitable, directly or indirectly detectable atom or molecule is meant. Suitable labels are, for example, fluorescent dyes and / or biotin and / or digoxigenin conjugated to nucleotides or polypeptides, such as antibodies,
And / or contains a radioisotope incorporated into a nucleotide or polypeptide, eg, an antibody. In a preferred embodiment, the labeling compound has a difference in the fluorescence behavior of the emission spectrum, such as for example coumarin and rhodamine, sufficient to select a small amount of substrate and / or for example fluorescein isothiocyanate and europium chelate Having a difference in fluorescence lifetime, such as labeled and porphyrin-labeled avidin;
It is a fluorescent dye.

According to a further preferred embodiment of the method according to the invention, the at least one reagent is capable of performing a hybridization or an antigen / antibody reaction or a ligand / protein reaction. The expression "hybridization", in particular "in situ hybridization", refers to a synthetic, naturally occurring DNA / RNA sequence in which a biological, physical or chemical label has been added as a reagent molecule for detection. DNA / RNA prepared
Refers to annealing of the probe, wherein annealing is achieved by denaturation and regeneration of the corresponding nucleic acid. Of course, these DNA / RNA probes can be used for DNA / RN of fixed biological material molecules such as chromosomes.
It contains at least one sequence portion capable of hybridizing with the A sequence. This sequence portion comprises a specific, individually occurring sequence range of a reagent molecule, preferably 100-1,000 base pairs in length, the reagent molecule comprising a complementary range of molecules of the immobilized biological material. At a suitable temperature, preferably up to 60 ° C., and 50 to 300 mmol / l of monovalent ions and 0 to 1
Anneal with hydrogen bridge formation at a suitable salt concentration preferably containing 0 mmol / l of divalent ions.

A further subject of the invention consists of a diagnostic kit for examining immobilized biological material according to one of the above-mentioned methods, said kit comprising a contact hybridization for immobilizing reagents thereon. A small area local cover, preferably comprising a microcover slip of

As an example of metaphase analysis, the method of the present invention uses an instrument for targeting and relocalizing suitable pre-localized metaphase chromosomes, so that it is simple, rapid and routine. It is performed so that it can be suitably applied as a work. The location of the metaphase chromosome, which has been localized manually or automatically, is stored. The localized metaphase chromosomes are then automatically positioned under a pipette using the probe solution to apply the hybridization solution in small quantities. In a further step, the localized metaphase chromosomes can be positioned under another pipette or suitable instrument to automatically apply a small cover slip.

For example, the consumption of substances in the hybridization of a metaphase chromosome with a DNA probe can be reduced by a factor of 10 or more with the method according to the invention and with the kit according to the invention. This is particularly important in tumor cytogenetics, where, on the one hand, the number of metaphases per microscope slide is low, and on the other hand, prior to DNA probe application,
Of particular interest is the possibility of metaphase pre-selection according to an embodiment of the present invention, for example, through the display of automatically localized metaphase photographs showing chromosome quality suitable for evaluation.

The method and diagnostic kit according to the present invention
Upon detection of minimal post-disease disease, is pre-labeled with a suitable marker and exhibits certain properties, such as tumor cells,
It can be equally applied to interphase cells that occur infrequently in a sample, or fetal cells in maternal blood.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 C12N 15/00 A (71) Applicant 599154168 Robert-Bosch-Strasse 6 D-68804 Altlusheim Germany (72) Inventor Andreas Plesch Germany D-68723 Schwetzingen Schellsigweg 78 (72) Inventor Oscar Ahr. Haas Austria Ar-3400 Kloster Neuburg Planckgasse 14

Claims (19)

[Claims] 1. A method for targeting and applying at least one reagent onto an immobilized biological material on a carrier, comprising: (a) localizing the immobilized biological material to be examined; Recording the position; (b) positioning the instrument for applying the reagent on the position of the immobilized biological material recorded in step (a); and (c) immobilizing the biological material. Applying a reagent onto the target material, wherein one or more locations of the same or different immobilized biological material on the carrier are recorded. 2. The localization of the immobilized biological material to be examined is performed in step (a) using a search optic, and in step (b) the search optic applies a reagent. The method of claim 1, wherein the method is replaced with a suitable device. 3. The method further comprises the step of: (d) positioning the device to apply the small area local cover over the location of the immobilized biological material whose location was recorded in step (a). The method according to claim 1 or 2, wherein 4. The method according to claim 1, wherein the application of the reagent is automated.
3. The method according to any one of 3. 5. The method according to claim 3, wherein the small-area topical cover is coated or moistened with a reagent. 6. The method according to claim 3, wherein the application of the small-area local cover is automated. 7. One of the localized and immobilized biological substances
7. The method according to claim 1, wherein several pictures are recorded during step (a). 8. The photographs are displayed together for pre-selection of a suitable fixed biological material.
The method described in. 9. The method according to claim 1, wherein the at least one intermediate step for treating, preferably washing, the immobilized biological material comprises:
localization of the fixed biological substance to be investigated in (a) and its location record, and at least one of the immobilized biological substance in step (b) on the location recorded in step (a) 9. A method according to any one of the preceding claims, wherein the method can be incorporated between positioning of a device for applying the reagent of the invention. 10. The evaluation is performed after step (c) or (d) by positioning the analytical device on the position of the immobilized biological material whose position was recorded in step (a). The method according to any one of claims 1 to 9. 11. The method according to claim 1, wherein the fixed biological material to be localized comprises cells. 12. The cell is labeled with a specific marker.
The method according to claim 11. 13. The method according to claim 1, wherein the fixed biological material to be localized comprises metaphase chromosomes. 14. The method according to claim 1, wherein the reagent comprises a nucleic acid selected from DNA or RNA, or comprises a polypeptide. 15. The method according to claim 1, wherein the reagent has one or several identical or different labels.
The method described in one. 16. The method of claim 15, wherein the label of the reagent comprises at least one fluorescent dye. 17. The method according to any one of claims 14 to 16, wherein the reagent is capable of performing a hybridization or an antigen / antibody reaction or a ligand / protein reaction. 18. The method according to claim 1, comprising a small area local cover for contact hybridization.
A diagnostic kit for analyzing immobilized biological material according to the method of any one of claims 14 to 17, wherein at least one reagent according to any one of claims 14 to 17 is immobilized on the cover. Kit. 19. The diagnostic kit according to claim 18, wherein the small area local cover is a microcover slip.